Tobacco treatment with citric acid and deuterium oxide

ABSTRACT

Treatment of tobacco to render smoking of the same safer. Reduction of the quantity of various undesirable constituents, particularly benzo(a)pyrene, in smoke resulting from the pyrolysis of such tobaccos in cigarettes and the like is shown as well as biological studies indicating such smoke condensates to be physiologically less dangerous. A mixture of citric acid and deuterium oxide is utilized to effect the foregoing modification of the combustion products of tobacco treated therewith.

United States Patent lnventor Max August Helmut Bindig Munich, Germany Appl. No. 809,004 Filed Mar. 20, 1969 Patented May 11, 1971 Assignee American Chemosol Corporation Priority July 16, 1965 Germany B82874 Continuation-impart of application Ser. No. 662,299, Aug. 22, 1967, now abandoned Continuation-impart of application Ser. No. 533,631, Mar. 1 l, 1966, now abandoned.

TOBACCO TREATMENT WITH CITRIC ACID AND DEUTERIUM OXIDE 12 Claims, No Drawings US. Cl 131/17, 131/140 Int. Cl A24b 15/02 Field of Search 131/17, 140l44 [56] References Cited UNlTED STATES PATENTS 2,971,517 2/1961 Pihl 131/140 3,003,895 10/1961 Grunwald... 131/17 3,106,210 10/1963 Reynolds et al. 131/17 3,112,755 12/1963 Parmele et a1 131/17X 3,126,011 3/1964 l-laden 131/17 3,380,458 4/1968 Touby et al 131/17 OTHER REFERENCES Tang & Mathur Deuterium lsotope Effects on Dissociation Constants and Formation Constants, from Journal of Physical Chemistry 1961) No. 65, pg. 1074- 1076 Primary Examiner-Melvin D, Rein Attorney-Holman & Stern TOBACCO TREATMENT WITH CITRIC ACID AND DEUTERIUM OXIDE This application is a continuation-in-part of copending application Ser. No. 662,299 filed Aug. 22, 1967, now abancloned which in turn is a continuation-in-part of application Ser. No. 533,631 filed Mar. 1 l, 1966, and now abandoned.

This invention relates to the treatment of tobacco and relates more particularly to a technique for processing tobacco in order to reduce the quantity of various undesirable constituents, particularly benzo(a)pyrene, in the smoke resulting from pyrolysis of a treated tobacco charge in smoking products, particularly cigarettes, manufactured with such tobacco compositions.

Since it is believed that a significant practical result of tobacco treatment according to this invention is diminished carcinogenicity in smoke therefrom, and to provide a better comprehension of the background of the instant invention, consideration should be given initially to certain factors, namely, (a) cancer as a disease, (b) those materials which, according to the present weight of authority, are believed to be carcinogenic, (c) the relationship between smoking and cancer as suggested by certain prominent members of the scientific community, (d) the various theories regarding carcinogenesis and (e) the generally accepted animal testing procedures for determining the effects of carcinogens and the effectiveness of cancer-reducing.techniques.

As indicated, this invention is concerned with the carcinogenicity of certain smoke constituents and thus, an understanding of materials believed to cause cancer and the activity thereof in connection with smoking is a basic prerequisite to an appreciation of the invention. Similarly, the significance of the results achieved with the invention are understood by comparison results with certain accepted testing procedures, and more particularly, rodent tests. Accordingly, the background on cancer-producing materials and cancer tests is set forth initially.

Cancer is a malignancy of pathologically changed cells which begins locally, grows in neighboring tissues and is carried throughout the body by the blood stream or lymph tract. Such pathogenic cells can form off-shoot tumors in remote regions of the body, a process known as metastasis. The entire organism can be thus affected depending upon the malignancy, location and size of such tumors.

The ability of certain chemical irritants, particularly with chronic reactions, to cause cancer has been well documented. Further, the presence of such carcinogenic materials in smoking means, particularly cigarettes, is now generally accepted. The Report of the Advisory Committee to the Surgeon General of the Public Health Service entitled Smoking and Health, Public Health Service Publication No. 1103 (hereinafter Surgeon General s Report"), a recent fully comprehensive study of the relationship between smoking and various diseases including cancer, clearly substantiates this fact.

Cigarette smoke is known to be a heterogeneous mixture of various materials including minute particles of liquid, uncondensed vapors and gases. When it reaches the smokers mouth, smoke is a concentrated aerosol with millions or billions of tiny particles, on the order of 0.5 microns in individual median size, per cubic centimeter.

Chemical composition and biological properties of smoke are ordinarily investigated by separating the same into a particulate phase and a gaseous phase, the gaseous phase frequently being further subdivided into materials which condense at liquid-air temperature and those which do not. The large quantities of material required for investigation of the chemical constituents are prepared on smoking machines in which many cigarettes are smoked simultaneously in a fashion designed to simulate average smoking habits. A yellow-brown condensate known as tobacco tar is collected in traps with the traps being cooled to the temperature of dry ice (-70 C.) or liquid nitrogen (196 C.). The tar thus contains all of the particulate phase of the smoke as well as condensable components of the gaseous phase. The amount of tar from the smoke of one cigarette is generally between 3 and 40 milligrams, the quantity varying according to the burning and condensing conditions, the length of the cigarette, the use of a filter, the porosity of the cigarette paper, the content of tobacco, and the weight and kind of tobacco.

Some 500 different compounds have been identified in either the particulate phase of cigarette smoke or in the gase ous phase. For example, components of the particulate phase include higher polycylic aromatics, aliphatic and alicyclic hydrocarbons, terpenes and isoprenoid hydrocarbons, alcohol and esters, sterols, aldehydes and ketones, acids, phenols and polyphenols, alkaloids, nitrogen bases, heterocyclics, amino acids and inorganic chemicals such as arsenic, potassium and some metals.

Many of the compounds found in cigarette smoke have not been shown to have carcinogenic activity. However, a variety of the polycyclic hydrocarbons and heterocyclic analogs have been found to produce cancer in mice and rats both by application to the skin and by subcutaneous injection, a testing procedure which, as will be pointed out in more detail hereinafter, is the conventional method for determining carcinogenicity and relating the same to the production of cancer in man.

The Surgeon Generals Report lists the following seven polycyclic compounds which have been isolated in cigarette smoke as having carcinogenic properties:

Compound Amount reported, Carclnoug./1,000

Structure geniclty cigarettes 1. Benzo(a)pyrene 2. D1benzo(a,i)pyrene Amount reported, Carcino- IJQJLOOO Compound Structure genicity cigarettes 3. Dibenzo(a,h)anthracene 4 4. Benzo(c)phenanthrene r. Q6] 4- 5. Dibenz(a,j)acridine \q 2.

6. Dibenz(a, h)acridine r i 0. 1

N 7. 7H-dibenzo(c, g) carbazoie 0.7

1 Not stated.

carcinogenic; +=Weakly carcinogenic.

The compound identified as No. 1 above, benzo(a)pyrene (occurs as l,2-benzopyrene; also called 3,4-benzpyrene and benz(e)pyrene, 4,5-benzopyrene is one of the most potent carcinogens detected in tobacco smoke and is present in much larger quantities than any of the other compounds identified to date. This material has been well known as a carcinogen for some time since it is present in coal tar, i.e., the tar formed on carbonization of coal.

Since the response of most human tissues to exogenous factors is similar qualitatively to that observed in experimental animals, it is believed to be highly probable that the tissues of man are also susceptible to the carcinogenic action of some of the same polycyclic aromatic hydrocarbons which have been found to cause cancer in test animais. The greatest consistency is observed in respect to those groups of chemical compounds which are carcinogenic in many animal species. Several of the polycyclic aromatic hydrocarbons present in tobacco smoke, particularly benzo(a)pyrene, fall into this category in that they are carcinogenic for most animai species tested.

0 found in tobacco smoke are, with perhaps one exception,

much less potent than benzo(a)pyrene and they are present in smaller amounts. Thus, there appears to be some synergistic effect in the production of carcinogenic activity.

Various materials, designated cocarcinogens in the Surgeon Generals Report, though not themselves carcinogenic, have been shown to enhance the cancer-producing properties of, or threshold to, known carcinogens.

Moreover, possible synergism between low levels of the several carcinogens in the tobacco condensates may enhance the carcinogenic potency.

Almost every species that has been adequately tested has proven to be susceptible to the effect of certain polycyclic aromatic hydrocarbons identified in cigarette smoke, particularly benzo(a)pyrene and designated as carcinogenic on the basis of tests in rodents. in fact, the Surgeon Generals Report indicates that the relatively high susceptibility of mouse skin to carcinogenic hydrocarbons has made it a favorite test object. Therefore, one can reasonably postulate that the same polycyclic hydrocarbons may also be carcinogenic in one or more tissues of man with which they come in contact.

Although the Surgeon Generals Report seems to indicate that the most significant health hazards are caused by cigarette smoking, in contrast to the use of other smoking means such as cigars or pipes, it is clear that various potentially carcinogenic materials are present in any tobacco smoke. The instant application is therefore directed to reduction of at least one element responsible for carcinogenicity in tobacco smoking means in general, as carcinogenicity is defined hereafter, or at least the reduction of the quantity of one material in smoke from the tobacco charge in any smoking device, which material is commonly accepted as a known carcinogen. However, for convenience, most references herein will be directed to cigarette smoking since this has created the most important medical problems.

To date, it would appear no practical method has been devised for eliminating or even for significantly reducing the suspected carcinogenic properties of cigarette smoke notwithstanding significant efforts to do so. Some attempts have been made to reduce the total smoke condensates, commonly known as tars, by mechanical filtration or by specific selection of particular tobaccos. Further, attempts have been made to incorporate into tobacco certain additives to produce a modified combustion and thereby reduce the production of materials in the smoke, believed to be carcinogenic, but such suggestions have generally not been practical from a commercial standpoint.

The continuous increase in cigarette production and use, even since the publication of the Surgeon Generals Report linking smoking with cancer and other possible diseases, evidences the fact that mere warnings on the part of prominent officials are insufficient to affect the smoking habits of most individuals. Therefore, it is extremely important that some means be devised to reduce the possible health hazards which result from smoking.

In this respect, although cancer has been discussed hereinabove as the primary consideration, it is well known that smoking has been associated with various other diseases such as various nonneoplastic respiratory diseases, particularly chronic bronchitis and pulmonary emphysema as well as various cardiovascular diseases. Coronary and peripheral arterial effects, combined with lung cancer, constitute the principal categories of human diseases related to cigarette smoking. The importance of modifying tobacco for use in cigarettes and the like to at least significantly reduce some of the welldocumented dangers to health is believed, therefore, to be apparent.

Considering the above background it is a primary object of this invention to provide tobacco and smoking means such as cigarettes made therefrom which present the likelihood of reduced dangers to the smoking public. Specifically, it is an object of this invention to provide a means for significantly reducing the quantity of benzo(a)pyrene present in the smoke resulting from pyrolysis of ordinary tobacco compositions since this material has been considered as an indicator of the carcinogenicity of tobacco smoke residues. The level of such reduction which is to be considered significant must be based on the biological efi'ects of the smoke condensates, a minimizing or eliminating of, for example, carcinoma resulting from testing such smoke condensates on conventional laboratory animals according to well acceptedscientific protocols obviously being an extremely significant result to the smoking public. From a practical standpoint, by reducing the quantity of benzo(a)pyrene in the smoke condensates, the presence of one of the two most potent of the seven carcinogens known in tobacco smoke will be diminished. Additionally, the wearcinogenic properties of other substances in smoke which may act synergistically with benzo(a)pyrene can be expected tobe reduced or avoided.

Based on the present state of knowledge, the smoke condensates from all smoked tobacco compositions, regardless of their source, include a quantity of benzo(a)pyrene which is detectable by well-known qualitative techniques and which is present in sufficient quantities to be a hazard to health as a tumor-inducing material. It has been suggested that the reduction of benzo(a)pyrene would inhibit the tumorigenicity of tobacco smoke condensate, benzo(a)pyrene being the accepted indicator for tumor-initiating aromatic hydrocarbons. See, for example, Reduction of Tumorigenicity of Cigarette Smoke, Wynder et al. JAMA, Vol. 192, No. 2, Apr. 12, 1965, pp. 96l02.

It should be recognized that the techniques to be described hereinafter are believed to significantly alter other of the combustion products of treated tobacco. However, due to the substantial time and expense required to study all phases of this problem, and the known significance of benzo(a)pyrene as a health hazard, the reduction of benzo(a)pyrene has been selected for detailed examination as a key to improved safety in smoking cigarettes and the like containing tobacco treated according to this invention.

From a practical standpoint, the basic consideration must be a modification of the biological effects of treated tobacco. Yet, reduction in quantity of known carcinogenic materials in smoke condensates can be viewed as indicating the likelihood of improved biological results. Therefore, chemical studies can be helpful in evaluating the effectiveness of the tobacco treatment hereof, although the specific biological tests discussed in some detail hereinafter cannot be overemphasized as the most important area to'be considered in determining the reduction in health hazards realized as a result of treating tobacco in accordance with the instant inventive concepts.

Although reducing the possible cancer-producing tendencies of cigarette smoke based on the present understanding of the properties of certain materials such as benzo(a)pyrene known to be in such smoke, is the basic and primary objective of the instant invention, it is also an important object hereof to reduce the possible death of tissues from arterial occlusion which may result from cigarette smoking as evidenced by certain commonly accepted tests in experimental animals. Further, it has been found that the techniques hereof significantly reduce the quantity of nicotine in tobacco smoke regardless of the brand oftobacco used, and this is believed to result in reducing the cough and irritation commonly resulting from cigarette smoking.

From a safety standpoint, this invention, in general, is directly concerned with the reduction of the biologically undesirable constituents of cigarette smoke without substituting any similarly noxious substances therefore, thereby in effect, leading to the production of a safer cigarette.

There are certain criteria other than functional effectiveness which must be satisfied if a procedure of the type with which the instant invention is concerned is to be accepted by the industry for use in the manufacture of tobacco products such as cigarettes and the like. Similarly, the smoking public is not likely to accept cigarettes or other similar smoking means which have been treated in a manner to reduce certain otherwise undesirable constituents in the smoke therefrom if the treatment also results in loss of the taste" characteristics which are considered desirable. Thus, it is another important and basic object of this invention to provide for the treatment of tobacco for use in smoking means such as cigarettes wherein the smoking means can be manufactured without unduly adding to the complexities and expenses normally encountered in present production techniques and without in any significant way deleteriously impairing the taste of the resultant products.

Consistent with the invention, the procedures for manufacturing cigarettes and the like having the improved properties set forth above do not result in taste losses, and require only relatively small additional capital investments for equipment. Further, the invention permits the utilization of particular compositions and techniques. the cost of which would not in any way be prohibitive to the adoption of such procedures by the industry. Additionally, it is an object of the instant invention to provide compositions for the treatment of tobacco, which compositions include only harmless ingredients not affecting metabolism in man or in animals. Specifically, the tobacco treating compositions hereof are nonresidual biologically and probably do not survive pyrolization in any form other than carbon dioxide and water. Further, the extremely small quantities utilized will not appreciably alter the weight of the tobacco.

Other and further objects of this invention reside in the specific chemical constituents and combinations of the same in the compositions hereinafter set forth as well as the particular manipulative steps of the various procedures utilized. Still other objects will in part be obvious and in part be pointed out as the description of the invention proceeds.

The basic techniques hereof comprise spraying a tobacco composition with a treating material comprising a solution of a mixture of effective quantities of citric acid and deuterium oxide. The following Example defines the preferred technique for carrying out the tobacco treating process of the instant invention:

EXAMPLE 1 TOBACCO TREATMENT Although distilled water has been used throughout the following procedure in order to avoid introduction of any unknown constituents, it is believed that de-ionized water or even a good grade of tap water can be readily substituted therefor without significantly affecting the results obtained. Preparation of stock solution:

One gram of citric acid was dissolved in distilled water to a total of milliliters and l milliliter of deuterium oxide was then added.

Dilution of stock solution for use:

0.2 milliliter of stock solution was diluted to l to 2 liters final volume with distilled water. Dilution with distilled water regularly gives a pH of 4.5 to 5.5 at between 1 and 2 liters final volume. It is found that within this pH range tobacco flavor and aroma are not appreciably altered.

Preparation of tobacco for spraying:

Shredded tobacco of conventional American blend ready for manufacture into cigarettes was used. Such tobacco ordinarily contains various flavoring ingredients and various humectants, depending on the manufacture. While the particular humectants may vary with different manufacturers, such additives are always included to provide the tobacco with adequate water retention properties for commercial purposes. Tobacco ready for manufacture into cigarettes generally has a relative humidity of approximately 50 percent plus or minus a few percent since unduly dry tobacco is generally not acceptable to the smoking public. In any event, the humidity of the tobacco should be adjusted to a range of about 48 or 50 percent to about 60 percent, if necessary, since the optimum results from use of the instant inventive concepts are realized by starting with tobacco of this type. Additionally, humidity control provides a simple measure for quantifying the treating solution added to the tobacco according hereto. Further, the relatively high humidity insures complete impregnation of the tobacco with the treating solution. The use of a humidity at this level causes no difficulty since, as indicated, ordinary tobacco ready for manufacture into cigarettes has a relative humidity of about 48 or 50 percent to satisfy other commercial requirements.

Spray technique:

Compressed inert gas, preferably nitrogen, was utilized as a propellant although oxygen or other propellants may be substituted therefor. A fine wetspray nozzle was employed with 4 atmospheres pressure of propellant gas. However, this is not a critical factor. As indicated, spraying tobacco with the aforementioned diluted solution was quantified by humidity control. The initial humidity was elevated by the spray liquid minimally to a reading 20 percent greater than the original humidity.

Temperature:

The entire procedure hereof was carried out at temperatures ranging from 2025Centigrade, that is, room temperature. The temperature requirements hereof are dictated by practical considerations and do not appear critical. The significant factor to note is that it is not necessary to provide any external heating means for the techniques hereof.

Although the preferred composition as set forth above comprised approximately a 10 percent citric acid and deuterium oxide solution diluted to a pH of about 5 and quantitatively controlled by humidity elevation, it has been found that other concentrations of solution and other quantity controls are useful. A composition in which 1 weight percent of deuterium oxide was added to a 10 weight percent citric acid aqueous solution which was then diluted with distilled water to a pH of about 5 was found to provide results similar to those to be reported hereinafter when 300 milliliters of such solution was sprayed on each kilograms of tobacco. This solution then has a weight ratio of about 1:10 deuterium oxide/citric acid as compared to a 1:1 solution as used in the preferred procedure described above.

In any event, it will be seen that the quantities of the active ingredients, that is, citric acid and deuterium oxide, are relatively minute whereby it would appear as if substantial variation in the actual quantities utilized should not be expected to significantly modify the resultant properties of the tobacco or the condensates resulting from smoking the same. For example, the ratio of citric acid to tobacco may vary as much as from 1 mg. to 100 mg. citric acid/tobacco for 5000 cigarettes. Since the average cigarette includes approximately 1 to 1.1 g. tobacco the citric acid is present in from about 1 mg. to 100 mg. to about 5 kg. tobacco. The deuterium oxide may be present in similar or even smaller proportions. Thus, the extremely small amounts of treating solution necessary to produce the desired results is believed evident.

As indicated, the solution described hereinabove can be prepared at ordinary room temperatures and is preferably applied to the tobacco as a spray. In actual production, conventional tobacco treating techniques are used except the solution is preferably sprayed onto the tobacco after the roasting or toasting operation at a location prior to the wrapping of the tobacco in the manufacture of a cigarette and at a point where the heated tobacco has sufficiently cooled so that it does not evaporate the spray solution. Generally, a location where the tobacco has reached a temperature of approximately 30 C. or less, that is, when the tobacco approaches room temperature, can be expected to be quite satisfactory. Substantially higher temperatures might evaporate the solution or make application of the same more difficult.

Spraying of the solution may be carried out in a flavor drum situated after the roasting equipment. Of course, other equivalent spraying equipment may be utilized in lieu of the flavor drum. It is to be pointed out that this spray equipment is basically the only additional investment which would be required for the use of the instant process whereby such a technique can be readily integrated with conventional cigarette manufacturing procedures in the absence of significant increase in costs.

Of course, with the preferred spraying technique, the particles should be of such a size that they would not tend to float or become suspended in the surrounding atmosphere. In other words, during the spraying technique, the particles should relatively quickly come in direct contact with the tobacco and impregnate the same.

As indicated, the solution is preferably used at a pH of between about 4.5 and 5.5. While this pH can be modified somewhat, a pH substantially below 4 will ordinarily adversely affect the taste and flavor of the treated tobacco. Therefore, this lower limit becomes somewhat important from a practical viewpoint. in fact, in order to produce optimum test characteristics, a pH of approximately is significant. Similarly, a maximum limit. of about pH 6 is basically governed by practical considerations in that a solution having less acidity would be undesirably weak whereby it would become necessary to add an excessive amount of the solution to the tobacco mixture thereby increasing the moisture content of the same to a point which is not commercially desirable.

It will accordingly be understood that although the procedure set forth in Example 1 is preferred from all standpoints, various modifications can be made thereto by those skilled in the art without departing from the instant inventive concepts. Additionally, it is quite possible that various chemically similar materials can be substituted for the ingredients of the composition set forth hereinabove while producing a similar result, although the time and expense in determining the available alternatives has not permitted such an investigation to date. However, it is now known that use of a aqueous citric acid solution alone or use of an aqueous deuterium oxide solution alone at least in the aforementioned concentrations rather than use of a mixed solution as set forth above will not produce the desirable modification of the smoke condensates to be discussed in more detail hereinafter, specifically, reduction of benzo(a)pyrene and health hazards. Although it may be possible that some other acid can be substituted for citric acid in the combination or some other deuterium-containing compound can be substituted for deuterium oxide in the combination, it does not appear that the combination can be modified by merely utilizing one or the other of the ingredients by itself.

EXAMPLE 2 CHEMICAL TESTS As mentioned in Example 1, ordinary commercial cigarette tobacco of "American" blend was utilized.

Utilizing conventional single cigarette, manually powered devices for manufacture of cigarettes, approximately 125,000 cigarettes were produced with tobacco treated according to the preferred technique of Example 1 and an additional 125,000 cigarettes were produced utilizing untreated tobacco of the same blend. Separate machines of identical construction were employed to make cigarettes from the treated and untreated tobaccos. Cigarettes were manufactured from each sample in identical fashion and with identical mean weights of tobacco. The cigarettes were 80 mm. overall, 63 mm. of which was tobacco, the remainder being a conventional cellulose acetate filter. Each cigarette weighted about 1 gram. The two batches of cigarettes were kept under identical conditions of temperature and humidity control.

Following manufacture, the 250,000 cigarettes were smoked in a conventional 40-port cigarette smoking machine. Cigarettes containing treated and untreated tobacco were smoked separately and the smoking machine was completely overhauled and cleaned between successive batches. The cigarettes were smoked utilizing the techniques commonly accepted for such procedures, that is,.a standard rate of one puff per minute of 2 seconds duration and 35 ml. volume, to within about 10 mm. of the filter. The smoke residues were collected on Cambridge fiberglass filters with no more than 5 residues being collected on any one Cambridge filter. The vapors which bypassed the Cambridge filters were trapped in flasks immersed in dry ice.

The Cambridge filter and cold trap residues were combined separately for the 125,000 treated and for the 125,000 untreated cigarettes. The,respectively separate residues were worked up in batches representing 500 cigarettes each.

For the chemical 'tests, the following bench procedures were followed with every batch:

l. The Cambridge filters were placed in 2 liter beaker.

2; With the aid of a large squeeze bottle containing methyl alcohol, glass delivery tubes were washed down in the cold traps. The cold traps were removed and dipped for a few seconds in a warm water bath. The methyl alcohol from the cold traps was poured into a beaker containing the Cambridge filters. The traps were then rinsed with methyl alcohol and the rinse added to the Cambridge filters.

3. One liter of methyl alcohol was added to the beakers with the Cambridge filters therein and allowed to soak thoroughly until the Cambridge filters disintegrated. A large ground glass pestle was useful to facilitate disintegration.

4. The mixture was poured into a Buchner funnel containing ordinary filter paper. Filtration was accomplished by suction with the aid of a water aspirator, into a three liter suction flask.

5. When filtration was complete, the packed filter pad was removed and resuspended in another liter of methyl alcohol.

6. The filtering operation was repeated. The methyl alcohol extracts from steps 4 and 5 were placed in a 3-liter separatory funnel. The funnel stopcock was lubricated with a few drops of water only and the stopper was held in place with an O ring.

7. The filter pad was resuspended in one liter of cyclohexane and filtered as before. This extract was then added to the separatory funnel for partitioning.

8. The mixture was shaken thoroughly. The pressure was released by inverting the funnel and slowly turning the stopcock. The phases were allowed to separate.

9. A long stem funnel was inserted through the top and through the upper cyclohexane layer into the methyl alcohol layer. About 2530 ml. of distilled water was slowly added and a light brown emulsion appeared at the bottom of the funnel.

10. The small funnel was removed and a long stirring rod was inserted which reached down into the emulsion. The material was stirred gently with a swirling motion. The brown color spread through the solution, but the phases separated sharply. This was allowed to stand for l hours.

1 l. The methyl alcohol layer was poured into a second funnel and the cyclohexane was poured into a round bottom flask of two liter capacity.

12. The cyclohexane was evaporated in a conventional spherical evaporating vessel wherein the vessel is rotated in a warm water bath to prevent uneven heating, the bath temperature being at about 56 C. Vacuum was produced by a conventional aspirator and was allowed to build up about one minute. The flask was then rotated, slowly at first to prevent bumping, then more rapidly.

13. Meanwhile the methyl alcohol fraction was extracted twice with 500 ml. of cyclohexane without any addition of water. If any emulsion formed at this point, the methyl alcohol layer was poured off and ml. cyclohexane was added to the emulsion. This was usually enough to break the emulsion.

' 14. The subsequent cyclohexane extracts were dried in the same flask as the first, and the same precautions were taken.

15. The residue was soluble in ether.

Final solubility of total residues in ethyl ether was accompanied by sulfuric acid removal of nicotine. Nothing else was extracted or otherwise removed. Ether solutions of residues were then dissolved in sesame oil (U.S.P.); the ethyl ether was blown off under vacuum and warm bath.

Utilizing conventional testing techniques, cigarettes formed with the tobacco treated according to the preferred technique of Example 1 were found to produce reduced nicotine content as compared with cigarettes incorporating untreated tobacco of the same blend, the treated condensates, on the average, containing approximately 78 percent of the nicotine in the untreated condensates, although total solids content was not significantly changed. In view of the known undesirable effects of nicotine, this reduction in nicotine content has obvious significance.

Extraction of polycyclic hydrocarbons from the smoke condensates and separations of the fractions to detect benzo(a)pyrene were done by standard chemical techniques:

1. Use of solvent pairs for partitioning.

2. Chromatography:

a. silica alumina c. thin-layer.

Location of areas in which benzo(a)pyrene might be located was done with black light." The suspicious areas were eluted and determinations for benzo(a)pyrene were made on each eluate. Spectrophotometric analysis of suspicious portions from this whole analytical system was performed.

Initial testing results consistently showed that smoke condensates from cigarettes containing tobacco treated according to Example 1 yield no detectable benzo(a)pyrene while, in each instance, tobacco smoke condensates from cigarettes containing the untreated tobacco provided positive identification of benzo(a)pyrene. Quantitative analyses of residues from cigarettes with untreated tobacco provided a minimum recovery, on the average, of about 1.2 gr of benzo(a)pyrene per 100 cigarettes, an amount which is comparable to that shown by other investigators. However, with the same techniques, no benzo(a)pyrene was found in residues of cigarettes having tobacco treated according hereto.

The initial testing procedures were based on the Grimmer method (Eine Methode zur Bestimmung von 3,4-Benzpyren in Tabakrauch Kondensaten von G. Grimmer, Biochemische Abeilung des Institutes fur Organische Chemie der Universitat Hamburg in the publication Beitrage zur Tabakforschung, Hamburg, No. 3, Aug. 1961, pages l071 l6) and were found to be insufficiently sensitive to detect the quantities of 3,4 benzo(a)pyrene present in condensates from cigarettes containing tobacco treated according to this invention. Recent testing has utilized the more sophisticated Davis et al. method (Analytical Chemistry, Vol. 38, p. 1752, 1966) which shows that benzo(a)pyrene is consistently reduced in quantity in smoke condensates from tobacco treated according to this invention as compared to untreated controls. The percent reduction as compared to the controls has been found to vary somewhat with the humidity level of the treated tobacco, but in substantially all instances some significant reduction has been realized regardless of the level to which the humidity was raised by addition of a treating solution according to Example 1. The following data is illustrative, all tobacco used in these tests initially having a relative humidity of about 48 percent.

The percent reduction is based on a comparison with the amount of 3,4 benzo(a)pyrene obtained from a control of the same tobacco composition:

TOBACCO A Percent Humidity after spray: reduction Contro 59 9. 65 17. 0 69 31. 0 70 39. 0 77 15. 0 85 14. 0 95 19. 0

TOBACCO B Control 64 3. 0 66 31. 0 68 28. 0 7O 32. 0 74 35. 0

The above shows that while the percent reduction varies with the final humidity level and the particular tobacco composition, some reduction in 3,4 benzo(a)pyrene is realized with each treated tobacco as compared with an untreated control of the same composition. The magnitude of the reduction appears optimum at from about 20 to 25 percent added humidity, this level also providing the best tobacco characteristics from the standpoint of subsequent processing of the same into cigarettes. An excessively large increase in humidity may cause difficulty in tobacco handling and seems to produce a drop in the benzo(a)pyrene reduction.

The initial humidity can also vary substantially while producing a benzo(a)pyrene reduction, but once again, optimum results are obtainable with tobacco having a conventional initial humidity level of approximately 50 percent.

Similarly, incorporation of conventional humectants in the tobacco composition is important to provide the optimum treatment according to this invention, such additives also being necessary for commercial production of cigarettes or the like from the tobacco in order to provide adequate water retention.

In summary, the most effective processing conditions according to this invention from the standpoint of temperature, humectant additives and initial an final humidity levels correspond with the most desirable conditions for commercial production of cigarettes whereby the techniques of this invention may be readily integrated with present production procedures.

The mechanism through which benzo(a)pyrene is reduced in smoke condensates from tobacco treated according hereto is not presently known However, it is clearly established that such treatment reduces the quantity of benzo(a)pyrene in cigarette smoke. Gas-liquid phase (GLC) identification and quantification chemistry confirmed that while benzo(a)pyrene was reduced in cigarette combustion products by the tobacco pretreatment hereof, it is also apparent that other noxious substances were not simultaneously increased.

The fact that benzo(a)pyrene is reduced in cigarette smoke as a consequence of the treatment set forth in Example 1, coupled with the known carcinogenic and cocarcinogenic effects of this material in cigarette smoke condensates, leads to the anticipation that the risk of cancer from cigarette smoke of treated tobacco would appear to be significant reduced by the techniques hereof. The biological studies set forth hereinafter fully confirm this expectation.

Another interesting phenomenon uncovered as a result of the testing program hereof was the fact that the burning temperature of tobacco treated in the manner of Example 1 is not significantly different from the burning temperature of untreated tobacco. Ordinarily, it is considered that fuel additives may produce modified combustion products by modifying the burning temperature of the tobacco, although the materials hereof have modified the combustion products at least by reducing the benzo(a)pyrene and nicotine content without significantly modifying the burning temperature.

Further, although total solids were not significantly quantitatively changed, chromatographic studies have shown modification of the combustion products from a qualitative standpoint even beyond the reduction of benzo(a)pyrene and nicotine mentioned previously. The specifics of these modifications have not as yet been studied in detail although it is known that other noxious substances have not been substituted for those eliminated or reduced. Exemplary of such modification in combustion products is a consistent overall reduction in the polycyclic hydrocarbon fraction even beyond the reduction in 3,4 benzo(a)pyrene. Moreover, there is a relative reduction of benzo(a)pyrene in the overall hydrocarbon fraction as compared with untreated controls as well as an absolute reduction in benzo(a)pyrene level on a per cigarette basis.

In human trials on about individuals who normally smoked a brand of commercially available cigarettes having the same blend of tobacco as was subjected to treatment according hereto, the treated cigarettes hereof were found to be at least as desirable from a taste and aroma standpoint to the smoker as their regular brand, without exception. Additionally, a reduction in cough and irritation was observed when smoking cigarettes having tobacco treated according hereto as compared with their regular brand. it is believed that the modification of the combustion products caused by the tobacco treatment hereof accounts for this reduction in cough and irritation.

EXAMPLE 3 BIOLOGICAL TESTS Total cigarette residues trapped in sesame oil were used for injection experiments. The only modification of the residues was removal of nicotine in all instances by conventional techniques since rodents are hypersensitive to nicotine poisoning and would be killed by nicotine present in smoke residues tested in quantities large enough to be significant.

The condensates were immediately taken into solution and prepared in the sesame oil for subcutaneous injection in the manner to be described in detail hereinafter. Smoking was continued throughout these biological tests and the aforedescribed chemical tests so that only fresh condensate would be used. Consequently, condensate was never stored in large quantities or for long times. In some instances small quantities of condensates were stored for short periods, never in excess of 48 hours, and such condensates were refrigerated during storage and warmed to ambient temperatures before use.

Experimental animals for these studies were CF-l (Carworth) inbred, tumor-resistent mice. Both males and females were used, selected at identical ages and average weights with the view to observing growth and weight-gain alterations.

These experiments were begun with all mice 21 days of age. The breeder and investigators were satisfied that the degree of inbreeding negated the need for splitting litters between the various groups to be described hereinafter.

In all groups injections were given just beneath the dermia of the mid-dorsal body skin with separate, disposable 25 gauge hypodermic needles and standard disposable tuberculin syringes. Conventional sterile precautions were observed.

Low-Dosage Experiments Of initial concern was the low-dosage" experiments.

A total of 150 mice were divided into three experimental groups, as follows:

Group A In this Group of 50 mice, each animal received 0.1 ml. injection of pure sesame oil from the batch used in Groups B and C each day for days (No tobacco smoke condensate was added).

Group B In this Group of 50 mice each animal received 0.1 ml. of smoke condensate (less nicotine) from five cigarettes containing tobacco treated according to Example 1 each day for 10 days.

Group C In this Group of 50 mice each animal received 0.1 ml. of smoke condensate (less nicotine) from five untreated cigarettes each day for 10 days.

These mice were examined daily. No noticeable changes occurred with all mice remaining healthy until the 14th day when approximately one-fifth of the mice of Group C were noted to have beginning ulceration at the injection site. The mice of Groups A and B appeared normal at that time. By the 20 day, one-fourth of the mice of Group C had ulcerations of injection sites with definite loss of hair. The mice of Groups A and B were normal 5 weeks from the start of the experiment.

Biopsies of the ulcerated areas and subsequent sloughs in animals receiving higher concentrations of cigarette concentrates showed microscopic changes of intense inflammatory reaction with vascular thrombosis and necrosis of all tissues in the area resulting in a slough of the tissues.

Biopsies of multilobulated masses occurring in all three Groups showed a temporary pseudocyst formation with no characteristic pathological change.

Experiments on Growth Rates The effects of low-dosage injection of cigarette smoke residues upon growth and the ability to thrive were studied by comparing the effects caused by residues of cigarettes containing tobacco treated according hereto and residues from the same brand of tobacco without such treatment.

The injected material in each Group of mice was the total smoke condensate (less nicotine) from five cigarettes. Ten

consecutive daily injections were given each animal. A body weight drop during the injection period was seen when the condensates from untreated tobacco was used. Reversibility occurred when the injections were stopped. The animal body weight injected with smoke condensates from tobacco treated according to the instant inventive concepts approximates that of sesame oil controls, that is, no body weight drop is seen.

High-Dosage Experiments High-dosage experiments were performed using injected total residues at three levels:

A. 25 cigarettes per injection for 10 consecutive days B. 50 cigarettes per injection for 10 consecutive days C. 100 cigarettes per injection for 10 consecutive days Sesame oil controls of equal injected volumes were established on all experiments). At each level, paired animals were injected with residues from tobacco treated according hereto and with untreated cigarette residues of the same brand. Observations on these mice are divided into two types:

I. Toxic mortality-4 weeks 2. Vascular impairment and local tissue destruction The toxic mortality from 25 cigarettes X 10 daily injection animals was zero for the sesame oil controls, the mice given residues from treated tobacco and those given untreated residues.

The toxic mortality from 50 cigarettes X 10 daily injection animals was zero for the sesame oil controls and the mice given residues from tobacco treated according hereto. From the third to the fifth week following the first injection 60 percent of the untreated residue mice died.

The toxic mortality from 100 cigarettes X 10 daily injection animals was 50 percent from both residue from treated and untreated tobacco.

Thus, treatment of tobacco according to this invention yields a decided decrease in toxic mortality in experimental animals over that which results from the residues of smoke from the same but untreated tobacco.

Vascular impairment and local tissue destruction from 25 cigarettes X 10 daily injection animals was zero in the sesame oil controls and the treated tobacco residue injected mice. The untreated residues produced vascular occlusion and tissue death at the injection site in 100 percent of the animals at the fourteenth to sixteenth day.

Vascular impairment and local tissue destruction from 50 cigarettes X 10 daily injection animals was percent in treated tobacco residue mice and percent in the untreated ones.

Vascular impairment and local tissue destruction from 100 cigarettes X 10 daily injection animals was 100 percent regardless of the treatment of the tobacco.

All of the substances from cigarette smoke which may cause vascular occlusion in man are not known. The type of tissue destruction seen in all these animals is typically and peculiarly that which results from arterial occlusion.

The lowest dosage level assayed does not produce arterial occlusion and tissue death, if the tobacco is treated according to the instant inventive concepts. This protective effective persists above the 25 cigarette per injection level. Protection provided by the treatment hereof against vascular impairment and tissue destruction is therefore of an order of magnitude of fiveto ten-fold.

Tumorigenicity The development of malignancy within 60 weeks in 21-day old mice was selected as the appraisal time desired. Such a program precludes dealing with the spontaneous malignancy of old mice.

Three groups of 100 mice each were treated in the manner of Groups A, B and C described above and were observed for a 60-week period.

Twenty animals (20 percent of original 100) developed malignant tumors in this Group C. Sixty percent of these were highly anaplastic fibrosarcoma. Note Tables, I, I1 and III hereinbelow. As will be seen, malignant tumors did not develop in these Groups A and B.

TABLE I.-300 CF; INBRED Y UN G MICE [GO-Week Experiment] 100 animals 100 animals 100 animals Total Total smoke smoke condencondensates sates 5 cigarettes cigarettes daily daily Sesame (treated (untreated all 0.1 tobacco) tobacco) Died, autopsy: no tumor i l 4 3 Died, causezmalignant tumor. 0 0 2 Living, known malignant tumor 0 0 10 1 2 of these were accidental traumatic deaths. 1 S fibrosarcoma 21ymph0sarcoma (2 more lympllosarcoma are dying) TABLE IL-AUTOPSY FINDINGS 18 ANIMALS Given the strain used, there is a difference in biological behavior of, for example, fibrosarcoma in young and in old mice. The former, when induced to produce this malignancy, have growths which may be transplantable within the strain; the latter do not. Three-fourths of the fibrosarcomas induced in the Group C mice were successfully transplanted through two generations within the CF strain.

All fibrosarcomas and apparently all induced lymphosarcoma are capable of causing death of the animals.

Skin Painting Experiments A popular brand of cigarette tobacco (U.S.A) was obtained as cut tobacco shreds. This tobacco was divided into two equal parts by weight, one half of which was treated according to the preferred technique of Example 1, the other half being untreated. By standard methods described elsewhere, these two batches of tobacco were used to fabricate treated cigarettes and untreated cigarettes using separate but identical machines. Mean weights of the two were identical.

A 40-port automatic smoking machine was used to smoke" both the treated and untreated cigarettes. Standardized, internationally accepted specifications for the smoking-volume of puff, time, interval, etc. were met. Total smoke condensates were collected in dry ice traps. No Cambridge filters were used. The whole condensate, without physical or chemical separation, was used. Condensates were collected in single glass containers for each 500 cigarettes smoked. They were refrigerated until use, at which time condensates were taken up in acetone for application to the skin. The smoking, the collection of smoke condensate and their application to skin all were in continuous operation throughout the experimental period. Equal numbers of treated and untreated cigarettes were smoked. The smoking machine was dismantled and new tubing and glassware substituted before each change from treated to untreated cigarettes.

The CF mice used in this experiment were housed in metal cages in air-conditioned (temperature/humidity forced air controlled), isolated rooms at 25C. without central air returns. No other experimental animals were permitted in the rooms used, and there was no change in laboratory personnel. Sawdust bedding and Purina Fox Chow Checkers ad lib, with water ad lib was provided. indirect daylight in equal intensity reached all cages; there was no exposure to bright sources of artificial light. All mice were 21 days old at the commencement of the experiments. All experimentscontrols, treated and untreated-were run concurrently for a 60-week experimental period.

A shaved area 1.5 cm. by 2.0 cm. in the mid-dorsal skin was selected for each animal. A 04 Camels hair brush was used to paint the whole condensate on this area. The volume of solvent (acetone) was not varied between controls and the experimental groups. All groups were painted on the same days, using different equipment. Excess condensates in acetone were discarded at the end of each days application.

Groups of 50 mice each were given, by dermal painting, the following applications three times weekly:

A. Total smoke condensates from 10 untreated cigarettes in acetone;

B. Total smoke condensates from 10 treated cigarettes in acetone; and

C. A volume of acetone equal to the volumes of B and C groups for each application.

The animals were examined once each day. All animals dying during the experiment were subjected to autopsy and pathological evaluation. Skin lesions developing in the painted area were observed, described, photographed and, if persistent and enlarging, biopsied. All biopsies were conducted under sterile surgical conditions. Permanent paraffin blocks were prepared after fixation, and microscopical slides were prepared and examined. All animals are accounted for during the experimental period. The date of death for animals not surviving 60 weeks was recorded.

Group C mice, which serve as overall controls, received only acetone, In this group there were no malignancies produced within the painted skin zone.

Group A mice, which received dermal paintings with untreated tobacco whole condensates, developed malignancies in 25 percent of the animals within the painted skin zone.

Group B mice, which received dermal paintings with treated tobacco whole condensates, developed no malignancies within the painted zone.

Survival figures for all groups were adequate to give firm statistical significance to these figures.

The obvious result of these critical biological studies is that the animals receiving no protection from tobacco treatment according hereto produced a high percentage of malignant, anaplastic tumors. The treated tobacco condensates produced no malignant tumors.

In summary, the objects and advantages realized with the instant inventive concepts can be listed as follows:

1. Although cost per se is not the most critical question, it is significant to note that the cost of the techniques and material hereof are sufficiently low that they can be readily set aside as a barrier to general use.

2. Additionally, no significant alteration in the acceptability of cigarettes manufactured utilizing tobacco treating techniques according hereto is found in habitual cigarette smokers.

3. The overall irritation from heavy (three or more packs daily) cigarette smoking resulting from the use of tobacco treated according to this invention is substantially reduced, especially the productive cough ordinarily induced in many subjects by cigarette smoking.

4. The instant inventive concepts require little or no equipment change and no significant additional steps in manufacture of cigarettes and the like.

5. The toxicity of the treating material hereof is not found to be any problem there being no metabolic residual matter and only reagent grade, naturally occuring substances being utilized.

6. One further significant area is the persistence of the effects of the treating techniques on the cigarette tobacco during shipment and storage prior to consumption. Tobacco treatment according hereto persists for a time exceeding normal shelf life of to days for cigarettes made and consumed in the US. Deliberately, less than optimum or usual storage conditions have been employed in these tests.

7. Yet another important area is that of evaluating both general and regional damage to tissues and the effects upon growth rates in experimental animals. Residues from cigarette tobacco treated according hereto are strikingly less damaging to living tissues than from untreated tobaccos of the same blend.

8. Finally, smoke condensates from tobacco treated according hereto have less 3,4 benzo(a)pyrene, a factor which is of extreme significance considering the known carcinogenic effect of this material, and biological tests confirm that such treated tobacco produces smoke condensates which can be expected to be significantly less hazardous to health, based on results in experimental animals.

From the above, it will be seen that the effectiveness of the tobacco treating techniques of this invention is clearly evidenced by significant reduction in the smoke therefrom of materials such as 3,4 benzo(a)pyrene and nicotine. The known physiological effects of such materials clearly suggest a reduction in health hazards from smoking cigarettes or the like in which the tobacco charge has been treated with effective quantities of a mixture of citric acid and deuterium oxide.

References herein to carcinogenicity of tobacco smoke in general are based on the many suggestions thereof in the scientific literature such as described in detail in the Surgeon General's Report. The indications herein that smoke from tobacco treated according to this invention can be expected to have reduced carcinogenicity and otherwise present less health hazards to the smoking public are to be understood as referring to the generally accepted correlation of such hazards with results obtained from experimental animal testing procedures following conventional protocol such as set forth hereinabove.

After reading the foregoing detailed description, it will be apparent that the objects set forth initially have beensuccessfully achieved. ACCORDINGLY,

lclaim:

l. A method of processing tobacco comprising treating the tobacco with a material including a mixture of citric acid and deuterium oxide in quantities sufficient to reduce the quantity of benzo(a)pyrene in smoke resulting from pyrolysis of the tobacco.

2. The method of claim 1 wherein said material is an aqueous solution having a pH of between about 4 and 6, said deuterium oxide being present in from about 10 to about percent by weight of said citric acid.

3. The method of claim 2 wherein said pH is about 5.

4. The method of claim 2 wherein said material, in aqueous solution, is sprayed onto said tobacco in quantities sufficient to impregnate said tobacco with said mixture.

5. The method of claim 1 wherein said material comprises an aqueous solution of about 1 gram of citric acid and 1 milliliter of deuterium oxide in 10 milliliters of water diluted to provide said solution with a pH of between about 4.5 and 5.5, further comprising the steps of initially adjusting said tobacco to a humidity of from about 50 to about 60 percent, an spraying said aqueous solution onto said tobacco in sufficient quantities to raise the humidity of said tobacco by at least about 20 percent.

6. The method of claim 5 wherein said spraying is effected at a temperature of about 2030 C.

7. A smoking article including a tobacco charge adapted to be ignited and smoked, the tobacco composition in said charge including a sufficient amount of a mixture of citric acid and deuterium oxide to reduce the benzo(a)pyrene content in smoke resulting from pyrolysis of the tobacco composition.

8. A tobacco composition comprising tobacco and a sufficient amount of a mixture of citric acid and deuterium oxide to reduce the benzo(a)pyrene content in smoke resulting from pyrolysis of the tobacco.

9. The method of claim 2 wherein said tobacco is treated with said material in amounts sufficient to provide from about 1 mg. to 100 mg. of citric acid to each 5 kg. of tobacco.

10. The smoking article of claim 7 wherein said mixture of citric acid and deuterium oxide is present in amounts sufficient to provide a ration of citric acid to tobacco of about 1 mg. to 100 mg. citric acid per 5 kg. of tobacco, and said deuterium oxide is present in from about 10 to 100 percent by weight of said citric acid.

11. A cigarette having a tobacco composition according to claim 8.

12. A tobacco composition according to claim 8 wherein said mixture of citric acid and deuterium oxide is present in amounts sufficient to provide a ratio of citric acid to tobacco of about 1 mg. to 100 mg. citric acid per 5 kg. tobacco, and said deuterium oxide is present in from about 10 to 100 percent by weight of said citric acid.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 7 7 Dated May 1]., 1971 lnventofls) Max August Helmut Bindig It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Cdumn 3, line 58, "4,5-benzopyrene" should read -4,5-benzopyrene) Column 5, line 27, cancel the hyphen. Column 7, line 49, "manufacture" should read -manufacturer--. Column 9, line 1, "test" should read -taste-,- line 19, "a" should read --an--: line 51, "weighted" should read ---weighed--; line 74, after "in" insert -a--. Column 10, line 37, "hours" should read --hour-.

Column 11, line 2 should read --b. alumina--; line 12, "yield" should read --yielded-. Column 12, line 15, "an" should read --and-; line 22, after "known" insert a period; line 36, "significant" should read significantly--. Column 13, line 56, "20" should read --20th-. Column 14, line 16, "Sesame" should read-(Sesame Column 15, line 52, cancel the hyphen. Column 16, line 6, "04" should read --#4-: line 32, the comma should be a period. Column 18, line 1 2, "an" should read --and; line 32, "ration" should read --ratio--.

Signed and sealed this 19th day of October 1971 (SEAL) Attest:

EDWARD M.F'LETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Acting Commissioner of Patents FORM PC4050 HO'GQ] uscowwoc moan-was I U 5, GOVE NMINT RINTING QIFIC! VINO-dob: 

2. The method of claim 1 wherein said material is an aqueous solution having a pH of between about 4 and 6, said deuterium oxide being present in from about 10 to about 100 percent by weight of said citric acid.
 3. The method of claim 2 wherein said pH is about
 5. 4. The method of claim 2 wherein said material, in aqueous solution, is sprayed onto said tobacco in quantities sufficient to impregnate said tobacco with said mixture.
 5. The method of claim 1 wherein said material comprises an aqueous solution of about 1 gram of citric acid and 1 milliliter of deuterium oxide in 10 milliliters of water diluted to provide said solution with a pH of between about 4.5 and 5.5, further comprising the steps of initially adjusting said tobacco to a humidity of from about 50 to about 60 percent, an spraying said aqueous solution onto said tobacco in sufficient quantities to raise the humidity of said tobacco by at least aBout 20 percent.
 6. The method of claim 5 wherein said spraying is effected at a temperature of about 20*- 30* C.
 7. A smoking article including a tobacco charge adapted to be ignited and smoked, the tobacco composition in said charge including a sufficient amount of a mixture of citric acid and deuterium oxide to reduce the benzo(a)pyrene content in smoke resulting from pyrolysis of the tobacco composition.
 8. A tobacco composition comprising tobacco and a sufficient amount of a mixture of citric acid and deuterium oxide to reduce the benzo(a)pyrene content in smoke resulting from pyrolysis of the tobacco.
 9. The method of claim 2 wherein said tobacco is treated with said material in amounts sufficient to provide from about 1 mg. to 100 mg. of citric acid to each 5 kg. of tobacco.
 10. The smoking article of claim 7 wherein said mixture of citric acid and deuterium oxide is present in amounts sufficient to provide a ration of citric acid to tobacco of about 1 mg. to 100 mg. citric acid per 5 kg. of tobacco, and said deuterium oxide is present in from about 10 to 100 percent by weight of said citric acid.
 11. A cigarette having a tobacco composition according to claim
 8. 12. A tobacco composition according to claim 8 wherein said mixture of citric acid and deuterium oxide is present in amounts sufficient to provide a ratio of citric acid to tobacco of about 1 mg. to 100 mg. citric acid per 5 kg. tobacco, and said deuterium oxide is present in from about 10 to 100 percent by weight of said citric acid. 